Materials and Reagents

DMSO (Sangon Biotech, catalog number: D0231 )
Dihydrorhodamine123 (Invitrogen, Molecular Probes^®, catalog number: D632 ) (see Recipes)
MitoTracker Deep Red FM (Invitrogen, Molecular Probes^®, catalog number: M22426 ) (see Recipes)
0.1 M sodium phosphate buffer (pH 7.0) (see Recipes)

Equipment

48-well plate
Razor blade
Slides
Centrifuge
Confocal laser scanning microscope (Zeiss, model: LSM 510 META )

Procedure

Cut fresh cassava storage roots into smaller segments about 5 x 5 x 0.5 mm in length, width and height by razor blade. The areas (Figure 1):
1. The center: vascular tissue found within the center of the root cross-section.
2. The middle: the tissue filled with storage cells with streaks of xylem throughout.
  
  Figure 1. Cassava storage root cross-section (Rickard, 1985). 1: periderm; 2: sclerenchyma; 3: parenchyma; 4: latex tubes; 5: cambium; 6: parenchyma (The middle); 7: xylem vessels (The middle); 8: xylem bundles (The center).
Immersed in the sodium phosphate buffer with Dihydrorhodamine123 or MitoTracker Deep Red FM, stain for 10 and 20 min, respectively in the dark at room temperature.
Afterwards, wash with sodium phosphate buffer once before viewing under the microscope. Use sodium phosphate buffer to mount the sample on slides.
Use a Zeiss LSM (Laser Scanning Microscope) 510 META Confocal with the capturing program LSM 510 equipped with a10x and 20x objective.
Allow the microscope and lasers to warm up for about 20 minutes before use.
The settings used for each stain are as follows:
1. Dihydrorhodamine123 excitation/emission 488/515 nm.
2. MitoTracker Deep Red FM excitation/emission 635/680 nm.
First use the visible spectrum to find the sample, after that change to fluorescence spectrum (Figure 2).

Figure 2. Fluorescence determination of PPD in cassava storage roots. a, xylem vessel; b, bundle sheath; Scale bar = 20 μm.

Recipes

0.1 M sodium phosphate buffer, pH 7.0 (100 ml)
Mix 61.5 ml 1 M K₂HPO₄and 38.5 ml 1 M KH₂PO₄
Filter sterilize (0.45 μm)
Store at 4 °C
Dihydrorhodamine123 working solution
1. 50 mM Storage solution
  10 mg Dihydrorhodamine123 with 577.4 μl DMSO
2. 50 μM Working solution
  Sodium phosphate buffer dilution
  Store at -70 °C
MitoTracker Deep Red FM
1. 1 mM Storage solution
  50 μg MitoTracker Deep Red FM with 91.98 μl DMSO
2. 250 nM Working solution
  Sodium phosphate buffer dilution
  Store at -70 °C

Acknowledgments

The protocol was mainly adapted from the publication Xu et al. (2013). This work was supported by grants from the National Natural Science Foundation of China (31271775), the National Basic Research Program (2010CB126605), the National High Technology Research and Development Program of China (2012AA101204), the Earmarked Fund for China Agriculture Research System (CARS-12-shzp) and the BioCassava Plus Program from the Bill & Melinda Gates Foundation.

References

Rickard J. E. (1985). Physiological deterioration in cassava roots. J Sci Food Agric 36: 167-176.
Xu, J., Duan, X., Yang, J., Beeching, J. R. and Zhang, P. (2013). Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots. Plant Physiol 161(3): 1517-1528.